The present invention relates in general to methods for attaching polynucleotides to supports and in particular to methods for attaching a nucleic acid hybridization probe to a solid support.
One characteristic property of nucleic acid, which forms the heritable material of all living organisms, is its ability to form sequence-specific hydrogen bonds (i.e., to hybridize) with a nucleic acid having a complementary nucleotide sequence. This ability of nucleic acids to hybridize with complementary strands of nucleic acid has been exploited in techniques generally called hybridization assays.
In a hybridization assay, a nucleic acid having a known sequence is used as a probe to search a sample for a "target" complementary sequence. Labelling of the hybrid formed by the probe and the target permits the detection and quantitation of complementary sequence.
Because all strains of a particular micro-organism share a genetic component in the form of nucleic acids susceptible to diagnosis by means of a hybridization assay, hybridization assays are valuable research and medical tools. Detection of specific target nucleic acids enables accurate diagnosis of bacterial, fungal and viral disease states in humans, animals and plants. Additionally, the ability to probe for a specific nucleotide sequence is of potential use in the identification and diagnosis of human genetic disorders.
In one type of hybridization assay, called solution hybridization, a labelled polynucleotide probe is added to a solution of a sample to be searched for a target nucleic acid. In order to ensure that both the probe and a target are in a single-stranded state suitable for hybridization, the sample and probe are heated in order to break (denature) the hydrogen bonds which are found between complementary strands of a double-stranded probe or target, or which are found within the secondary structure of a single-stranded probe or target. Upon cooling, the reaction is reversed and double-stranded nucleic acid is allowed to form. The amount of double-stranded nucleic acid which forms may be determined by scintillation counting of the label on the probe after degradation of unhybridized single strands or after isolating double-stranded DNA by passing the hybridization solution over a hydroxyapatite column which selectively retains the double-stranded form. However, if either the probe or the target was introduced in double-stranded form, a reaction reforming (renaturing) double-stranded probe or a double-stranded target competes with the hybridization reaction between probe and target and thereby reduces the sensitivity of this technique.
In another approach to hybridization assays, renaturation is prevented by immobilizing denatured target nucleic acid on a support. After passage of the support-bound target through a solution containing labelled probe, retention of the probe on the support-bound target permits detection and quantitation of the target by measurement of the amount of bound label. See, e.g., Falkow, et al., U.S. Pat. No. 4,358,535; and Shafritz, European Patent Application No. Al-0062286. However, because the amount of labelled probe is far in excess of the amount of target present, non-specific binding of the labelled probe to the support may swamp the detectable signal from a small amount of target.
Still another approach to hybridization assays is called a "sandwich" hybridization. A two-step sandwich hybridization procedure involves the use of an immobilized target nucleic acid which is exposed in a first step to a first nucleic acid probe having a first portion complementary to the target and having a second portion which is not complementary to the target. In a second step, a second, labelled nucleic acid probe which is complementary to the second portion of the first probe is allowed to hybridize to the first probe, forming a "sandwich" with the first probe between the target and the second probe. Dunn, et al., Cell, 12: 23-36 (1977). The sandwich hybridization procedure is relatively easy to perform and is not seriously affected by protein or other biological contaminants. Ranki, et al., Gene, 21 77-85 (1983). However, a two-step sandwich hybridization assay involves considerable delay associated with immobilization of the sample on a filter.
It is neither easy nor convenient to attach a single-stranded nucleic acid probe directly to a solid support for use in a sandwich hybridization assay. For example, the attachment of a nucleic acid to a nitrocellulose sheet involves fixing the nucleic acid by contact with the sheet for 12-15 hours and baking the nucleic acid onto the sheet for two hours. See, e.g., Thomas, Proc. Natl. Acad. Sci. (USA), 77: 5201 (1980). Such preparation of a DNA-coated nitrocellulose sheet may easily consume as much as a full working day, a factor which limits the clinical usefulness of nucleic acid hybridization.
Furthermore, because the nucleic acid probe is sequence-specific for a particular target molecule, the procedure for attaching the probe to the support must be performed for each target molecule to be detected. Thus, in order to detect a number of different DNA sequences, a diagnostic laboratory must prepare an equal number of types of supports.